A combined mechanical and X-ray diffraction study of stretch potentiation in single frog muscle fibres

Abstract

The nature of the force (T) response during and after steady lengthening has been investigated in tetanized single muscle fibres from Rana temporaria (4 C; 2.15 micrometer sarcomere length) by determining both the intensity of the third order myosin meridional X-ray reflection (IM3) and the stiffness (e) of a selected population of sarcomeres within the fibre. With respect to the value at the isometric tetanus plateau (To), IM3 was depressed to 0.67 +/- 0.04 during steady lengthening at approximately 160 nm s(-1) (T approximately 1.7) and recovered to 0.86 +/- 0.05 during the 250 ms period of after-stretch potentiation following the rapid decay of force at the end of lengthening (T approximately 1.3); under the same conditions stiffness increased to 1.25 +/- 0.02 and to 1.12 +/- 0.03, respectively. After subtraction of the contribution of myofilaments to the half-sarcomere compliance, stiffness measurements indicated that (1) during lengthening the cross-bridge number rises to 1.8 times the original isometric value and the average degree of cross-bridge strain is similar to that induced by the force-generating process in isometric conditions (2.3 nm), and (2) after-stretch potentiation is explained by a residual larger cross-bridge number. Structural data are compatible with mechanical data if the axial dispersion of attached heads is doubled during steady lengthening and recovers half-way towards the original isometric value during after-stretch potentiation.

Figure 2. Axial and radial intensity distributions of the M3 reflection

A, superimposed intensity distributions along the meridian in the region of the M3 reflection: ○, rest; □, isometric tetanus plateau; ▵, lengthening; ⋄, after lengthening. The integrated area is ±1/85 nm⁻¹ across the meridian, according to the width of the linear detector. Two fibres from March 1999 visit. B, intensity distribution of the M3 reflection in the radial direction. The integrated area is from 1/12.5 nm⁻¹ to 1/17.5 nm⁻¹. In these fibres (6 fibres from March 1999 visit) values for W/W_r were 1.96 for the isometric tetanus plateau, 2.54 during stretch and 2.54 after stretch.

Figure 1. Record showing the experimental protocol

From top to bottom the traces represent: the change in length imposed at the end of the fibre connected to the motor, the change in the sarcomere length recorded by the striation follower (hs, half-sarcomere), force, the four periods when X-ray data were acquired, stimulus. The vertical bars below the force trace mark the times when, in stiffness experiments, length steps were imposed on the fibre. The times of the first (isometric plateau), second (during stretch) and fourth (after stretch) bar occur midway through the corresponding X-ray frames.

Figure 3. Tension transient in the three conditions investigated

A, force response to either step stretch (upper row) or release (lower row) of ≈1.3 nm half-sarcomere⁻¹ imposed at the isometric tetanus plateau (left column), during steady lengthening (central column) and 175 ms after the end of lengthening (right column). Fibre length, 6.36 mm; segment length, 1.30 mm; sarcomere length at rest, 2.07 μm; CSA, 13700 μm²; T₀, 250 kN m⁻². B, T₁ relations from another fibre in the three conditions in A. ○, isometric tetanus; □, lengthening; ▵, after lengthening. T₁ values are relative to T₀. Continuous lines are the linear regressions to each set of points; the slope of each line is the stiffness of the half-sarcomere (e, T₀ nm⁻¹). Fibre length, 6.10 mm; segment length, 1.05 mm; sarcomere length at rest, 2.09 μm; CSA, 23400 μm²; T₀, 310 kN m⁻².

Figure 4. Stiffness in the three conditions investigated

Mean changes (4 fibres, the vertical bars showing ±s.e.m. are within the size of the symbols) of relative force (T/T₀, •) and stiffness (e/e₀, ○) versus the time after the end of lengthening. Force is that just before the stiffness measurements. A continuous line is drawn for ordinate 1. The points at zero time are the values during steady lengthening. The dashed lines are the exponential fit to the three points during the after-stretch phase with the equation y = aexp(-kt) +c, where k (2.5 s⁻¹) is the rate constant estimated in Colomo et al. (1989) for the slow process, y is either the calculated T/T₀ or the calculated e/e₀, c is the calculated asymptotic value, and a is the amplitude of the exponential process.

Figure 5. Conformation of the myosin head at the isometric tetanus plateau (A) and during lengthening (B and C)

A, the myosin head (light grey) is in the isometric tetanus plateau conformation: the axial coordinate (z) of the tip of the lever arm (residue 843 in Rayment et al. 1993), marked with a dot, is 7.2 nm away from the rigor value (Dobbie et al. 1998). B, the myosin head (light grey) is in the average conformation assumed during steady lengthening: the lever arm is further tilted by 16 deg away from rigor orientation (z shifted by further 2 nm away from Z-line). C, case with both heads attached during lengthening: it is assumed that the lever arm of the first head (light grey) is back tilted by 32 deg from the isometric configuration and has promoted the attachment of the second head (dark grey, same tilting of the lever arm as isometric) on the actin site farther from the Z-line.